Development of a New Device to Combine Local Optical Stimulation and Mea Recordings

INTRODUCTION Since the introduction of the Micro-Electrode Array (MEA) technology [1], it has been exploited as a powerful tool providing distributed information about learning, memory and information processing in cultured neuronal networks, changing the field of view from single cell level to the scale of complex biological networks. Despite the great advantage of the MEA technology in recording extracellular activity, its applicability to cell culture/tissue stimulation presents some important limits related to the use of electrical stimulation in a conductive volume. Its major limits are the presence of large stimulus artefacts and the poorly controlled spread of electrical stimuli in the medium. Although some of the problems of stimulus artefacts have been recently solved using blanking circuits [2] and algorithms [3], the spreading of electrical signals remains a limitation of MEA technology. In fact, it has been demonstrated that electrical stimuli spread to the whole biological preparation with an amplitude decreasing with the square of the distance from stimulation site [4]. To overcome these limitations affecting the MEA-based electrophysiological analysis of network properties, alternative approaches based on optical technologies can be investigated as tools for the stimulation of neurons. In the last two decades several methods to couple light and neuronal excitation have been proposed [5], but above all the use of caged compounds seems to be the most physiological approach for the coupling of light with either neuronal excitation, e.g. with caged neurotransmitters, or modulation, e.g. with caged intracellular secondary messengers. Here we present the use of MEA biochips coupled to the local optical uncaging in order to obtain high localised stimulation at single electrical recording sites while monitoring the overall preparation. Moreover, we describe a new technological improvement, called PhotoMEA, aimed to integrate in the MEA biochip optical waveguides to address directly the UV light on the neuronal cultures in a very localised manner. METHODS Cell cultures. Low-density primary cultures of hippocampal neurons were prepared from embryonic day 18 rat embryos, essentially as previously described [7]. Hippocampi were dissociated by a 15min incubation with 0.25% trypsin at 37°C, and cells were plated at a density of 450-550cells/mm 2 on poly-L-lysine (1mg/ml)-treated devices in MEM, supplemented with 10% horse serum, 2mM glutamine , and 3.3mM glucose. After allowing neurons to adhere to the surface for 3-4hrs, a coverslip with astrocytes was turned upside down, with astrocytes facing down towards neurons. Physical contact between neurons and glial cells was avoided …